The present invention relates to methods of use and compositions for inhibiting endothelial cell tube formation, the initial step of tumor angiogenesis, and angiogenesis-dependent diseases in tissue, animals and humans. More particularly, the present invention relates to a tripeptide, its analogues, mimetics and chemical derivatives that show inhibition of angiogenesis-mediated processes such as cancer, ocular neovascularization, and inflammatory diseases. Anti-angiogenesis agents disclosed can be also used in conjunction with surgery, chemotherapy, radiotherapy, and laser therapy.
Angiogenesis is the development of new blood vessels from preexisting blood vessels (Mousa, S. A., In Angiogenesis Inhibitors and Stimulators: Potential Therapeutic Implications, Landes Bioscience, Georgetown, Tex.; Chapter 1, (2000)). Physiologically, angiogenesis ensures proper development of mature organisms, prepares the womb for egg implantation and plays a key role in wound healing. On the other hand, angiogenesis supports the pathological conditions associated with a number of disease states such as cancer, inflammation and ocular diseases.
The development of vascular networks during embryogenesis or normal and pathological angiogenesis depends on growth factors and cellular interactions with the extracellular matrix (Breier et al., Trends in Cell Biology 6:454-456 (1996); Folkman, Nature Medicine 1:27-31 (1995); Risau, Nature 386:671-674 (1997)). Blood vessels arise during embryogenesis by two processes: vasculogenesis and angiogenesis (Blood et al., Bioch. Biophys. Acta 1032:89-118 (1990)). Vascular endothelial growth factor (VEGF), bFGF, IL-8 and TNF-a are some of the growth factors that play a role in pathological angiogenesis associated with solid tumors, diabetic retinopathy and rheumatoid arthritis (Folkman et al., Science 235:442-447 (1987)). Angiogenesis is generally absent in adult or mature tissues, although it does occur in wound healing and in embryogenesis (Moses et al., Science 248:1408-1410 (1990)).
Angiogenesis or xe2x80x9cneovascularizationxe2x80x9d is a multi-step process controlled by the balance of pro- and anti-angiogenic factors. The latter stages of this process involve proliferation and the organization of endothelial cells (EC) into tube-like structures. Growth factors such as FGF2 and VEGF are thought to be key players in promoting endothelial cell growth and differentiation. The endothelial cell is the pivotal component of the angiogenic process and responds to many cytokines through its cell surface receptors and intracellular signaling mechanisms. Endothelial cells in culture are capable of forming tube-like structures that possess lumens. Therefore, endothelial cells are not only a prerequisite for neovascularization, but appear to be the basal structural requirement as well.
Angiogenesis-dependent diseases include the following: inflammatory disorders such as immune and non-immune inflammation, rheumatoid arthritis, psoriasis; ocular disorders such as diabetic retinopathy, neovascular glaucoma, retinopathy of prematurity, age-related macular degeneration, corneal graft rejection; and cancer associated disorders such as solid tumors, tumor metastases, and blood born tumors such as leukemia, angiofibroma, kaposi sarcoma, benign tumors, as well as other cancers, which require neovascularization to support tumor growth.
It has been proposed that inhibition of angiogenesis would be a useful therapy for restricting tumor growth. Inhibition of angiogenesis can be achieved by inhibiting endothelial cell response to angiogenic stimuli as suggested by Folkman et al., (Cancer Biology 3:89-96 (1992)), where it described examples of those endothelial cell response inhibitors such as angiostatic steroids, fungal derived products such fumagilin, platelet factor 4, thrombospondin, alpha-interferon, vitamin D analogs and D-penicillamine. For additional proposed inhibitors of angiogenesis, see Blood et. al., Bioch. Biophys. Acta 1032:89-118 (1990); Moses et al., Science 248:1408-1410 (1990); and U.S. Pat. Nos. 5,092,885, 5,112,946, 5,192,744 and 5,202,352.
In 1997, Kefalides and co-workers at the Connective Tissue Research Institute of the University of Pennsylvania, Department of Medicine, reported that the peptide corresponding to the residue sequence 185-203 of the noncollagenous 1 (NC1) domain of the xcex13-chain of basement membrane collagen type IV inhibited the activation of polymorphonuclear leukocytes (PMN""s) (Han et al., J. Biol. Chem. 272:20395-20401 (1997)). It was found that the peptide xcex13(IV) residues 185-203 having the sequence CNYYSNSYSFWLASLNPER (SEQ ID NO:1) promoted adhesion of human melanoma cells by 50-60% over controls and also inhibit their proliferation by 40%. Alanine substitution through the peptide sequence indicated that the observed activities were dependent on the presence of residues 189-191, referred to as the SNS sequence. The Kefalides group later reported the inhibition of melanoma cell proliferation by type IV collagen requires increased levels of cAMP (Shahan et al., Connective Tissue Res. 40:221-232 (1999)), the identification of CD47/integrin-associated protein (IAP) and xcex1vxcex23 as two receptors for the xcex13(IV) chain of type IV collagen on melanoma and prostrate cells (Shahan et al., Cancer Res., 59:4584-4590 (1999)). More recently, they have reported the Ca2+ dependency in tumor cell chemotaxis (Shahan et al., J. Biol. Chem. 275:4796-4802 (2000)) as well as the inhibition of expression and activation of matrix metalloproteinase by the NC1 domain of type IV collagen (Pasco et al., Cancer Res. 60:467-473 (2000)).
Independently, Kalluri""s group from the Harvard Medical School has also reported the characterization of the two different types of anti-tumor activities (anti-proliferation and anti-angiogenic) of xcex13(IV) NC1 domain using both in vitro and in vivo assays (Maeshima et al., J. Biol. Chem. 275:21340-21348 (2000)). Collectively, these reports effectively highlight the distinct and unique anti-tumor properties of the xcex13(IV) NC1 domain and its potential use as a lead for small molecule, anti-tumor drug design.
Absent from these reported results is the identification of a smaller recognition epitope that retains the activities of the larger peptides. The problem to be solved, therefore, is to provide the identification of a small recognition epitope which would be the crucial step in providing a template for structure-based drug design strategies towards small molecule analogues or peptidomimetics. Such small molecules would include cyclic peptides and peptide isosteres with preferable physiochemical and pharmacokinetic properties that intervene in angiogenesis-dependent diseases such as cancer.
The invention is directed toward an angiogenesis-inhibitory tripeptide of formula aa1-aa2-aa3, having a first amino acid (aa1), a second amino acid (aa2) and a third amino acid (aa3), wherein,
(a) said first amino acid is selected from the group consisting of Ser, Thr, Ala, Phe, Tyr, Cys, Gly, Leu, Lys, Pro, Arg, Gln, Glu, Asp, Asn, His, Met, Ile, Trp, Val, diaminoproprionic acid and trans-4-hydroxy-proline;
(b) said second amino acid is selected from the group consisting of Asn, Ala, Gly, Asp, Glu, Gln diaminoproprionic acid and trans-4-hydroxy-proline; and
(c) said third amino acid is selected from the group consisting of Ser, Thr, Ala, Phe, Tyr, Cys, Gly, Leu, Lys, Pro, Arg, Gln, Glu, Asp, Asn, his, met, Ile, Trp, Val, diaminoproprionic acid and trans-4-hydroxy-proline.
Methods of inhibiting angiogenesis by administering the tripeptide are also provided.